Homogenous assay for enzymatic activity

ABSTRACT

An assay is disclosed for measuring activity of enzymes, such as kinases, phosphatases, and proteases. Measurements of enzymatic activity are accomplished in a homogenous assay format utilizing a fluorescence quenching technique employing paramagnetic metal ions.

FIELD OF INVENTION

The present invention relates to the assay of enzymatic activity and,more particularly, to the measurement of the activity of kinases,phosphatases, and proteases, as well as other enzymes. Morespecifically, the present invention relates to measurements of enzymaticactivity, which are accomplished in a homogenous assay format utilizinga fluorescence quenching technique employing paramagnetic metal ions.

BACKGROUND OF INVENTION

Enzymes are catalytic proteins that typically act on a substrate toyield an endproduct. Protein kinases and protein phosphatases comprise aclass of enzymes that modify protein and/or peptide substrates bycatalyzing the attachment or removal, respectively, of a phosphorylgroup to sites on certain amino acid side chains of the substrates forthese enzymes. Determining the presence or catalytic activity of theseenzymes is important since the degree of phosphorylation of a particularprotein or peptide has been found to be an important characteristic inregulating cellular functions. Enzymes capable of peptide bond cleavage,known as proteases, are another class of enzymes. Ever increasingemphasis is being placed on discovering what drugs can be used tomodulate enzyme activity. This, in turn, has created the demand for thedevelopment of improved techniques for the measurement of the activityof enzymes.

Radioactive detection has been used to assay for enzymatic activity;see, for example, U.S. Pat. No. 5,538,858. With respect to kinases, asample containing the kinase of interest is incubated with activatorsand a peptide substrate in the presence of radioactive labeled ATP.Then, an aliquot of the incubated mixture (containing phosphorylated andnon-phosphorylated peptides) is placed on a filter that binds thesubstrate and the filter washed to remove excess radioactivity. Theamount of radiolabeled phosphate incorporated into the substrate and, inturn, enzyme activity is measured by scintillation counting.

Because of the necessity for the precautions involved in radioactivetechniques, non-radioactive assay techniques are also in use. Aparticularly attractive assay is sold by Pierce Biotechnology, Inc.(formerly known as Pierce Chemical Company) under the SpinZyme™ brandname and is described in U.S. Pat. No. 5,527,688, issued on Jun. 18,1996. In this assay, non-radioactive ATP is used in the incubationmixture and the substrate is dye-labeled. After the enzymatic reaction,the incubated mixture is brought into contact with a solid phasecontaining immobilized Fe⁺⁺⁺. The phosphorylated substrate in themixture binds to the solid phase by chelation with the iron ion. Thenon-phosphorylated substrate is removed by washing, and the amount ofphosphorylated substrate is measured by detection of the dye, which islabeled on the substrate.

The above-described assay techniques are non-homogenous in that theyrequire the phosphorylated and non-phosphorylated substrates to bephysically separated between the kinase-initiated phosphorylation anddetection. These added steps detract from the use of these techniques inthose applications commonly used in drug screening and termed highthroughput screening.

Homogenous assay techniques have been developed in a variety of specificareas to overcome the aforementioned drawbacks of non-homogenous assays.One example is radioactive assays, commonly known as scintillationproximity assays. These are described in U.S. Pat. Nos. 4,568,649,5,665,562 and 5,989,854. Other homogenous assays are described in theApril, 2002 issue of Drug Discovery & Development entitled “The Key toKinases is All in the Kits,” beginning on page 28.

Several other categories of homogenous assays are based onnon-radioactive detection methods. Fluorescence techniques, such asbased on fluorescence resonance energy transfer (FRET) and fluorescencepolarization (FP), have been introduced; see, for example, U.S. Pat. No.6,287,774 and US Patent Application 2002/0034766 A1.

In FRET methods, a fluorophore (a light-absorbing dye capable offluorescence emission) is utilized in combination with anotherfluorophore (either identical or not) or with a chromophore (alight-absorbing dye not capable of fluorescence emission). A generalrequirement for FRET is that the two entities of the pair combination(either fluorophore and fluorophore, or fluorophore and chromophore)have an overlapping spectral region. The ability of the FRET techniqueto be utilized in the construction of an assay relies on the capacity todistinguish, by measurable signal detection, the variation in observedfluorescent emission from the combination pairs employed when they arein close proximity as opposed to spatially separated. Thus, in thistechnique, a donor fluorophore, such as fluorescein, can be used withthe dye, tetramethylrhodamine, as an acceptor fluorophore. When thesetwo fluorophores are in close proximity to each other, excitation of thefluorescein molecule results in energy being transferred to thetetramethylrhodamine acceptor and consequently the normal expectedemission from the fluorescein is decreased.

Assays can be constructed using FRET techniques where specific bindingevents are utilized to bring the two fluorophores into close proximity.Such assays can be quantitated by observing decreased fluorescentemission of the donor fluorophore or by observing increased fluorescentemission of the acceptor fluorophore, both of which are brought aboutwhen the binding event occurs. Proteolytic FRET assays utilize theaction of a protease to cleave the substrate having the attachedfluorophores (for example labeled on the N and C terminus of peptide) tocause the two fluorophores to be more spatially separated and therebydiminishing the FRET event.

In the case of FRET assays utilizing a chromophore, examples of usefulchromophores include those commonly known as Black Hole Quenchers andDABCYL (4-(4′-dimethylaminophenylazo)benzoic acid) (See Proc. Natl.Acad. Sci. USA. 1999 May 25; 96 (11): 6394-6399 entitled “Multiplexdetection of four pathogenic retroviruses using molecular beacons.”Jacqueline A. M. Vet, Arnit R. Majithia, Salvatore A. E. Marras, SanjayTyagi, Syamalima Dube, Bernard J. Poiesz, and Fred Russell Kramer).

Drawbacks of the FRET technique include the requirement of a matchedcombination pair, which precludes a more universal utility to assayconstruction, along with other drawbacks, such as increased cost,complications related to assay interpretation via signal breakthrough,and negative assay interactions, such as hydrophobic interactions of theenzyme with the dye molecules, etc.

Turning now to the FP method mentioned above, this technique relies ondetecting a measurable change in fluorescent polarization. For example,in FP-based kinase assay, these assays measure the change in fluorescentpolarization (FP) that accompanies the kinase catalyzed phosphorylationof a fluorescent dye-labeled substrate. To achieve a measurable changein FP on phosphorylation, a large entity is included in the incubationmixture, which complexes with the phosphoryl group on the derivatizedpeptides. In such a direct assay format, because of the resultingincrease in size of those peptides that have been phosphorylated, theirrotational diffusion is significantly less and, in turn, their FPsignificantly greater, than of those peptides that have not beenphosphorylated. Competitive FP formats also have been described. Thus,the difference in FP between the labeled substrate before catalysis withthe kinase of interest and after catalysis is indicative of the activityof the enzyme.

FP assays can be run in a homogenous format, which requires no washingand separation steps because both the before and after measurements areof the same parameter, namely, fluorescent polarization; only the changein this parameter is the determinative factor. However, a drawbackassociated with FP assays is the necessity for using expensive equipmentcapable of measuring FP. A further drawback of FP assays resides incertain technical limitations associated with its use, such as assayartifacts due to scattered light, viscosity changes, and polarizationchanges associated with incorporation of small molecular weightfluorophores into large molecular detergent micelles. These limitationsare not found in fluorescent assays based on simple measurement offluorescent intensity.

SUMMARY OF INVENTION

In accordance with the present invention there is provided a method andassociated composition of matter for assaying the activity of an enzymebased on fluorescence quenching. The present invention results from theobservation that the fluorescent intensity of a fluorophore label on anenzymatic substrate or endproduct can be quenched by the presence of aparamagnetic metal ion when bound to a target group located in proximityto the label, and the realization that useful enzyme assays are enabledutilizing this observation.

The assays incorporate a paramagnetic multivalent ion that bindsspecifically to a target group present on a fluorescent dye-labeledenzymatic substrate or endproduct. When bound to the target group, theion is brought into proximity to the fluorophore and acts as a quencherof the fluorescent dye label by means of intrinsic properties of themetal ion. This specific binding is immediate. Results are quantitatedby comparing the observed relative fluorescence units of test samples toblanks containing no enzyme.

More specifically, the method of the present invention involves assayingthe activity of an enzyme of choice by contacting the enzyme with apopulation of fluorophore labeled substrate in an aqueous enzymaticreaction mixture, and allowing the enzymatic reaction to proceed for aselected period of time and temperature as desired. The reaction is thenbrought into contact with a paramagnetic metal ion to form a complex ofthe paramagnetic metal ion with a target group on either the enzymesubstrate or endproduct. This complex, when in proximity to thefluorophore label, causes specific quenching of the fluorescence fromthe fluorophore. By measuring the intensity of the observed fluorescentemission from the mixture and relating the observed fluorescence to thatof an external reference, a differential fluorescent signal, if any, canbe identified and quantitated to ascribe a specific value to thedifferential. This ascribed differential value in fluorescent signal ofthe sample is indicative of the final state (i.e., post-enzymaticreaction) of the fluorophore labeled substrate population and, in turn,reflects enzymatic activity.

Assays using the present invention can be practiced in several formats.In one embodiment the enzyme is reacted with a substrate to produce anendproduct containing a target group having binding affinity for theparamagnetic metal ion. In another embodiment, the enzyme reactionremoves such target group from the substrate. In both instances, thesubstrate contains an attached fluorophore label.

The method of this invention is particularly applicable in the assay ofkinase and phosphatase activity, including kinases and phosphataseacting upon peptide substrates. In both of these cases the target groupis a phosphoryl group. The endproduct is the fluorophore labeledsubstrate, which has either been phosphorylated in the case of kinase ordephosphorylated in the case of phosphatase. In the kinase assay, afteraddition of the paramagnetic metal ion to the endproduct, enzymeactivity is evidenced by decreased fluorescent intensity with increasingenzymatic activity. The decrease in fluorescent intensity arises as aconsequence of the increased content of peptide moieties containingphosphoryl groups in the overall population of fluorophore labeledpeptide present in the final enzyme reaction mixture.

With respect to phosphatase enzyme assays, by virtue of the fluorophorelabeled substrate being already phosphorylated, the initial startingenzymatic reaction will be more highly quenched prior to the enzymaticreaction. Thus, the observed fluorescent emission from the populationafter enzymatic removal of phosphoryl groups will be observed toincrease with increasing phosphatase activity since the action of theenzyme will result in a decrease in the overall population offluorophore labeled peptides containing the attached phosphoryl groups.

As can be seen from the two prior cases, the actual assay can result ineither a decrease or increase in fluorescence. It should be noted,however, that, in both cases, the mechanism relies on a specificquenching caused by paramagnetic metal ion with its target group.

Protease activity also can be assayed by the method of the presentinvention. In this case, a protease substrate is selected to have aproteolytic cleavage site between the attached fluorophore on thesubstrate and a target group, which can be an imidazole or phosphorylgroup. As such the target group resides in proximity to the label and,upon binding, the paramagnetic metal to the target group, the metal ionis consequently brought into proximity to the fluorophore label. As withthe case of the above mentioned phosphatase assays, the protease assaywill be observed to exhibit an increase in observed fluorescent emissionwith an increase in protease activity. This is because the protease willcleave the substrate to result in a population having fluorophore labelswith increased spatial separation as compared to the initial substratewhere the label is in close proximity to the paramagnetic metalion/target group complex.

Another aspect of the present invention resides in providing acomposition which is formed in the fluorescence quench-based homogenousassays for enzymatic activity described above. This compositioncomprises a paramagnetic metal ion and a substrate for an enzyme or anenzymatic endproduct resulting from reaction of the enzyme with asubstrate. The substrate or endproduct contains a fluorophore label andalso contains a target group to which the paramagnetic metal ion isbound, thus permitting the formation a complex of the target and ion.The complex is in proximity to the fluorophore and causes specificquenching of the fluorescence of the label when the complex forms.

Yet a further embodiment of this invention provides a kit comprised of aparamagnetic metal ion and an instruction booklet referencing and/ordescribing the manner in which the assay can be accomplished withrespect to one or more enzymes as set forth herein. The kit may includea synthetically prepared calibrator to function as an externalreference. The calibrator includes a fluorophore labeled syntheticcompound having one or more concentrations of the attached target groupof interest, each in separate packages.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the assay for PKC enzyme activity using three differentenzymatic substrates labeled with Lissamine rhodamine and conducted in a384 well format using an opaque black plate. The data represent themeans of triplicates and error bars show the standard deviation. Theobserved fluorescent signal decreases with increasing amounts ofenzymatic activity.

FIG. 2 shows the assay for PKC enzyme activity using three differentenzymatic substrates labeled with Lissamine rhodamine and conducted in a384 well format using an opaque white plate. The data represent themeans of triplicates and error bars show the standard deviation. Theobserved fluorescent signal decreases with increasing amounts ofenzymatic activity.

FIG. 3 shows the assay for PKA enzyme using Lissamine rhodamine labeledKemptide and conducted in either a 96 or 384 well format using opaquewhite plates. The data represent the means of triplicates and error barsshow the standard deviation. The observed fluorescent signal decreaseswith increasing amounts of enzymatic activity. The graph illustrates theraw observed signal as mean RFU or as the normalized signal expressed asthe mean percentage of RFU relative to the zero enzyme point assigned to100% fluorescence.

FIG. 4 shows the assay for PKC enzymatic activity using Lissaminerhodamine labeled pseudosubstrate activity conducted in either a 96, 384or 1536 well formats using opaque white plates. The graph illustratesthe normalized fluorescent signal expressed relative to the zero enzymepoint assigned a relative value of 1.0.

FIG. 5 shows the assay for tyrosine kinase activity using Lissaminerhodamine labeled TK1 peptide where the enzyme was allowed to react foreither one or two hours in a 96 well opaque white plate. The datarepresent the means of triplicates and error bars show the standarddeviation. The observed fluorescent signal decreases with increasingamounts of enzymatic activity. The observed fluorescent signal is morehighly quenched at each enzyme concentration with two hours of enzymaticreaction as compared to one hour of enzymatic reaction time, confirmingthe time-dependency of the enzymatic reaction.

FIG. 6 shows the inhibition of PKC activity by staurosporine. Theobserved fluorescent signal is differentially quenched relative to theamount of inhibitor supplied to the enzyme reaction mixture. The degreeof observed fluorescent correlates to the amount of enzymatic activity.The enzymatic activity is higher at lower concentrations of inhibitor,and therefore the observed RFU is decreased.

FIG. 7 shows the inhibition of PKA activity by PKI. The observedfluorescent signal is differentially quenched relative to the amount ofinhibitor supplied to the enzyme reaction mixture. The degree ofobserved fluorescent correlates to the amount of enzymatic activity. Theenzymatic activity is higher at lower concentrations of inhibitor, andtherefore the observed RFU is decreased.

FIG. 8 shows the assay for PP2A phosphatase activity using Lissaminerhodamine labeled peptide substrate LRRApSLG at one or two hours ofenzymatic reaction in a 96 well plate format. The data represent themeans of triplicates and error bars show the standard deviation. Theobserved fluorescent signal increases with increasing amounts ofenzymatic activity. The observed fluorescent signal is less quenched ateach enzyme concentration with two hours of enzymatic reaction ascompared to one hour of enzymatic reaction time, confirming thetime-dependency of the enzymatic reaction.

FIG. 9 shows the assay for PTPB or PTP1B phosphatase activity usingLissamine rhodamine labeled peptide substrate using the phosphorylatedTK1 peptide in a 96 well plate format. The data represent the means oftriplicates and error bars show the standard deviation. The observedfluorescent signal increases with increasing amounts of enzymaticactivity.

FIG. 10 shows the assay for inhibition of PP2A activity by okadaic acid.The observed fluorescent signal is differentially quenched relative tothe amount of inhibitor supplied to the enzyme reaction mixture. Thedata are normalized by assigning the observed RFU to equal 100% enzymeinhibition at the highest concentration of inhibitor tested.

FIG. 11 shows the assay for inhibition of PTP1B activity by sodiumortho-vanadate. The observed fluorescent signal is differentiallyquenched relative to the amount of inhibitor supplied to the enzymereaction mixture. The data are normalized by assigning the observed RFUto equal 100% enzyme inhibition at the highest concentration ofinhibitor tested.

FIGS. 12 and 13 shows the assay for proteolytic enzymatic activity ofTPCK-treated trypsin using the Lissamine labeled peptide substratesLRRApSLG or AGLARAGLALARLALALRRApSL, respectively. The observedfluorescence increases with increasing enzymatic activity, indicatingproteolytic cleavage at a peptide bond N-terminal to the phosphoserineresidue.

FIG. 14 shows an assay for protein phosphatase activity using eitherphosphatase PP-2Cα or PP2A. The phosphatases were incubated withrhodamine-labeled phosphorylated Kemptide as the enzyme substrate.

FIG. 15 shows the assay for PKA activity using fluorescein labeledKemptide substrates.

FIG. 16 shows the assay for PKA activity using Oregon Green labeledKemptide substrates.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an improved method for assaying thecatalytic activity of enzymes in a homogenous assay format. Asparticularly illustrated herein, but without limitation thereto, themethod is useful for measuring catalytic activity of (a) kinases inphosphorylating peptide substrates, (b) phosphatases indephosphorylating peptide substrates, and (c) proteases in cleavingpeptide substrates. The method relies on 1) the ability of aparamagnetic metal ion to exhibit specific affinity interaction with atarget group on a fluorophore labeled enzyme substrate or endproduct and2) the ability of the paramagnetic metal ion, when so bound, to quenchspecifically and preferentially the fluorescent emission of the dyelabeled enzyme substrate or endproduct. The concurrent use of these twofeatures together permits the construction of useful enzymatic assays. Arequirement of the assay is the inclusion of the fluorophore as a labelto enable the quench-based assay of the instant invention, and in mostinstances a single fluorophore will be the only dye moiety labeled onthe substrate.

As used herein, fluorescence quenching (or quenching) refers to areduction in the observed fluorescent emission intensity from a parentfluorophore relative to its intrinsic or expected fluorescent character.Many of the known characteristics associated with fluorescence quenchingand assays that rely on a quenching mechanism, and fluorescenceprinciples in general, are described in Principles of FluorescenceSpectroscopy, 2nd edition, Joseph R. Lakowicz, Kluwer Academic/PlenumPublishers, New York, 1999. A homogenous assay for determining enzymaticactivity, based on fluorescent intensity, employing a quenchingmechanism requiring only a single dye molecule distinguishes the quenchbased assay of this invention from other available quench assays,including those based on FRET, and is accompanied by the advantagesdescribed herein.

As indicated above, an important aspect of this invention is the use ofa paramagnetic metal ion having the ability to complex with a targetgroup on the substrate or endproduct and thereby effect specificquenching of fluorescence of a label thereon. As used herein, a“paramagnetic” ion is an ion having unpaired electrons as indicated in“Advanced Inorganic Chemistry, 4th ed.,” p. 1359, by F. A. Cotton and G.Wilkinson.

Metal ions useful in this invention are those that are paramagnetic andhave been recognized as having utility in immobilized affinity columnchromatography where their specific binding capacity has been used forpurification purposes. Particularly useful paramagnetic ions includeFe⁺⁺⁺ and Ni⁺⁺ (also identified herein as ferric ion or Fe (III), or Ni(II)). Fe⁺⁺⁺ exhibits specific binding to phosphoryl groups. Thus, inthese assays the phosphoryl is the target group for binding to theparamagnetic metal ion to form a complex. Accordingly, Fe⁺⁺⁺ isespecially useful in enzymatic assays involved with phosphorylation ordephosphorylation of substrates. Ni⁺⁺ finds particular utility due toits capacity for specific binding the imidizole target group onhistidine and polyhistidine tags. Thus, it is particularly useful inprotease assays, where the enzyme substrate incorporates these groups.

To achieve formation of the complex of ion and target, the ion cansimply be brought into contact with a fluorescent labeled substrate orendproduct in an aqueous incubation mixture derived from a simple saltsuch as FeCl₃ or NiCl₂. It is convenient to contact the paramagneticmetal ion with the substrate or endproduct in a pre-prepared aqueousform coordinated with a chelator. Such chelation improves handlingcharacteristics, solubility, and aqueous stability of the paramagneticmetal ion. Representative chelators for Fe⁺⁺⁺ and Ni⁺⁺ are iminodiaceticacid (IDA), ethylenediaminetetraacetic acid (EDTA), nitrilotriaceticacid (NTA), or salts thereof, as well as forms where the chelator isattached to a carrier molecule, such as a protein. Salts of paramagneticmetal ions presented to the enzyme substrate or endproduct as simplechelates are preferable by allowing the delivery of higher amounts ofthe paramagnetic metal ion to the incubation mixture for overcoming theimpact of interfering compounds. The utilization of multipleparamagnetic metal ions chelated to a carrier molecule can be employedwhere it is desired to increase the local molar ratio of theparamagnetic metal ion that can be bound in proximity to the fluorophorewhen the paramagnetic metal ion binds to its target group.

In practicing this invention, the amount of the paramagnetic metal ionadded, in a working solution, to the incubation mixture should besufficient to provide enough material to bind fully the target groups onthe peptide in the mixture. For practical applications, a slight excessis typically used. A slight excess of metal ion improves the functionalaspects of the assay with respect to reproducibility and robustness.Where there may be interfering compounds, such as in the case ofphosphorylation kinase assays, due to the presence of material in theincubation mixture capable of also binding Fe⁺⁺⁺, such as ATP and itsbyproduct, ADP, a larger excess can be used to increase further thereproducibility and robustness of the assay. With the above factors inmind, consideration also should be given to the fact that theparamagnetic metal ion can function as a collisional quencher, andthereby reduce the total fluorescent signal available for detection.Consequently, the optimum concentration of the paramagnetic metal is aconcentration providing the maximum differential fluorescent signal andwill be dependent on the labeled peptide concentration, along with theinstrumentation and format desired to conduct the assays. Table 1presented hereafter illustrates useful concentrations of theparamagnetic metal ion as presented within the context of a workingsolution for the typical conditions encountered in plate assays. Theseconcentrations were utilized in examples presented hereafter unlessotherwise indicated.

The paramagnetic metal ion can be delivered to the incubation mixturewithin a buffering solution, which serves to maintain the pH of thefinal solution (the solution prior to fluorescent measurement). Theparamagnetic metal ion also may be delivered to the incubation mixturewith additives, such as detergents and solvents.

With respect to pH, a final pH for the solution is selected that avoidspotential precipitation of any of the components, while maintainingappropriate pH to allow for specific binding of the paramagnetic metalion to its target group. In cases where ferric ion is utilized inphosphorylation/dephosphorylation enzyme assays, the utilization ofacidic pH values are useful and serve the added function of facilitatingstopping the enzymatic reaction in the incubation mixture. The additionof the paramagnetic metal ion can also facilitate stopping of theenzymatic reaction by other mechanisms. For example, in the case ofkinase assays, the addition of the ferric ion can facilitate thetermination of enzymatic activity by binding to the phosphoryl group ofATP and thereby making the ATP unavailable for enzymatic reaction.

Regarding the ability of the bound paramagnetic metal ion to effectspecific quenching, the ion when bound to the target is in proximity tothe fluorescent dye label. This spatial requirement can be met bychoosing an enzyme substrate or enzyme endproduct where the target groupfor binding the paramagnetic metal ion is less than about 50 amino acidsin length removed from the fluorophore. For practical purposes a spatialseparation of less than about 25 amino acids and generally 2 to 10 aminoacids is considered useful. However, it should be recognized that thespatial separation of a given labeled peptide in a linear primarystructure can differ from the actual spatial separation in solution, forexample, due to its secondary and tertiary structure in solution.

In practicing the assay of this invention, customary enzymatic reactionconditions are carried out to allow for the desired conversion ofsubstrate to endproduct. The addition of the paramagnetic metal ioncauses the metal ion to bind specifically to a portion of the totalinitial dye-labeled peptide substrate population. This specific bindingevent distinguishes enzymatic activity by allowing for discrimination ofthe enzymatically altered portion of the substrate in relation to thatportion of the substrate not altered, or not acted upon, by the enzyme.In practicing the invention, the observed fluorescent emission of theunknown sample can be compared to that of an external reference. Thereference can be a control, calibrator, or standard curve, which isoptionally predetermined.

The assay of this invention utilizes simple instrumentation. Because theassay is homogenous, no separation steps are required. Additionally, theonly instrumentation required to practice this invention is aconventional fluorometer or plate reader. There is no requirement in theassay for measuring changes in polarization signal as is the case in anFP assay and, therefore, no necessity for instrumentation to measure FP.Furthermore, using this assay, many technical limitations associatedwith other assays are minimized. The instant invention enables auniversal approach to assay construction through the use of a singlefluorophore, while reducing assay costs and simplifying assay design anddata interpretation.

The present invention can be practiced in any of the usual enzymaticreaction formats. Thus, a dye labeled peptide substrate is firstprepared, being selected according to desired specificity for the enzymeof choice. The labeled peptide may utilize any of the recognizedfluorophores as the dye. Examples of dyes considered to be usefulinclude Lissamine Rhodamine, BODIPY (Molecular Probes, Inc., Eugene,Oreg.), fluorescein, and Oregon Green. Other examples of fluorescentdyes are other dyes supplied by Molecular Probes, as well as thosefluorescent dyes manufactured by Amersham and Dyomics and others.Preparation of the dye labeled peptide substrate is accomplished bycommonly known procedures. For example, attachment of the fluorophore tothe peptide sequence is conveniently accomplished during peptidesynthesis by reaction of the N-terminus amino group of the peptide withthe dye. A nucleophilic derivative of the fluorophore, such as asulfonyl chloride derivative, may be utilized to effect the covalentattachment of the fluorophore to the N-terminal alpha amino group of thepeptide. However, other methods of attachment and at other locations canbe utilized.

Turning specifically to the practice of the present invention withrespect to the measurement of kinase activity, a population ofdye-labeled peptide substrate is initially phosphorylated inconventional fashion in an incubation mixture (i.e., the enzyme reactioncocktail). The incubation mixture is prepared in a buffer, such as Trisor HEPES. The mixture includes, as essential constituents, the proteinkinase of interest (generally serine/threonine kinases or tyrosinekinases), a source of high energy phosphate group such as ATP, anddye-labeled peptide substrate. It may also include other additives, suchas enzyme cofactors (e.g., Ca⁺² and Mg⁺²) for enhancing activity,activators for the enzyme (e.g., phosphatidyl-L-serine for conventionalprotein kinase C (PKC) isoforms and cyclic AMP for PKA). The mixture canbe further supplemented with common enzyme stabilizing agents, such as areducing agent (i.e., dithiothreitol) or a detergent (i.e., TritonX-100). In many assays, particularly for drug screening, putativeinhibitors or enhancers to be evaluated as to their influence on enzymeactivity are present.

The mixture, as above prepared, is allowed to incubate for a selectedperiod of time, generally about 10 minutes to 3 hours, to achieve thatdegree of phosphorylation of the substrate population indicative of theactivity of the enzyme in the fashioned environment of the incubationmixture. At this point, the enzyme reaction mixture contains apopulation of dye-labeled phosphorylated endproduct andnon-phosphorylated substrate. Then, in accordance with the presentinvention, ferric ion (Fe⁺⁺⁺) is added to the incubation mixture tosupply the paramagnetic metal ion. After addition of this ion, therelative fluorescence units (RFU) are measured on a fluorometer. Themeasured RFU value will be less than that observed for a control sampletreated in like fashion but without enzymatic turnover. This decrease isdue to the substantially complete quenching by Fe⁺⁺⁺ of the fluorescentsignal from the dye labeled on the phosphorylated peptides in thepopulation, whereas the non-phosphorylated peptide is not quenched tothe same degree. A comparison of the decreased fluorescent emission ofthis mixture with that of the control is a measure of enzyme activityfor a given assay; a larger decrease in fluorescent signal indicatingmore phosphorylation and, in turn, enhanced enzyme activity.

In practicing the invention with respective to determination of proteinphosphatase activity, conditions as described above for the kinaseenzyme activity can be employed with modifications chosen to reflect therequirements for enzymatic activity of the selected protein phosphatase.ATP, for example, can be omitted. In contrast to the observed assayresponse of a decrease in observed fluorescence with increasingenzymatic activity, the observed fluorescence will be increased withincreased enzymatic activity of the selected protein phosphatase. Bydefinition, the protein phosphatase will require the use of aphosphorylated dye labeled peptide substrate, and consequently theparamagnetic metal ion will bind essentially completely to the startingsubstrate as opposed to the enzymatic endproduct.

Turning now to the protease assay, the selected peptide sequencecontains a proteolytic cleavage site selected according to the proteaseof choice. The proteolytic cleavage site sequence is specifically chosento reside within the total amino acid sequence of the starting substratebetween the dye and the specific binding site for the paramagnetic metalbinding. If the paramagnetic metal selected for the assay is ferric ion,the binding site can, as with kinases and phosphatases be aphosphorylated amino acid. If the paramagnetic metal selected for theassay is divalent nickel ion, the binding site can be histidine orpolyhistidine. With respect to the enzymatic reaction cocktail andconditions required for enzymatic activity, the conditions are selectedaccording to the requirements for the chosen protease, and will betypically conducted in a buffered aqueous solution.

A requirement for the assay is that substrate chosen and the dye-labeledon the substrate allow the enzymatic reaction to proceed. A variety ofpeptide substrates, as well as conditions for their reaction, forkinases, phosphatases, and proteases are known from the literature thatallow for the assay of particular enzymes of choice. Considerationshould be given to the fact that modification of the peptide sequencewith the fluorophore may influence the nature of the enzymatic reactionas compared to that of a non-labeled substrate.

The selection of the peptide sequences to be utilized in theparamagnetic metal ion based assay of the instant invention, are chosenwith the following aspects in mind: 1) the peptide should remain solublein solution after addition of the paramagnetic metal ion, and 2) the“core” structure of the enzyme substrate or endproduct (i.e., thatportion not containing the target of the paramagnetic metal ion forspecific binding) should not have the propensity for significantnon-specific binding of the paramagnetic metal ion.

When a variety of specific substrates are available for investigation ofuse within the assay, the substrate providing the highest preferentialor differential quenching between the enzyme substrate and enzymeendproduct forms will allow for the highest detection sensitivity withrespect to enzymatic conversion. The differential response of the enzymesubstrate and endproduct forms can be easily determined. For example, inthe case of kinase assays, this can be accomplished by first obtaining asample of the phosphorylated and non-phosphorylated peptides and thendiluting them separately to low and essentially equal concentrations toprovide for substantially identical fluorescence emission afterexcitation at a chosen wavelength. Preferably, the concentration of thefluorophore should provide an absorbance at the excitation wavelengthsufficiently low to avoid inner filter effects. The Fe⁺⁺⁺ reagent isadded in sequential and identical increments and the fluorescenceemission is captured after each addition. The emission intensity of thefluorophore labeled peptides vs. amount of added reagent is thencompared; obtaining differential and preferential quenching of thephosphorylated form as compared to the non-phosphorylated form.Examination of the data dye-labeled illustrates the ability todistinguish, and thus provide an assay for, the two forms of thepeptide. The peptide pair yielding the highest differential responseidentifies the peptide pair providing the highest detection sensitivity.

The assay can be practiced in a variety of solution phase formats, suchas in cuvettes or microwell plates; the latter being particularlyemployed where high throughput capability is required. As to microwellplate format densities (i.e., 96 to 1536 well formats), each of theseformats have an upper assay volume limit dictated by the well capacityof each plate type. Higher density plates typically have smaller wellvolume capacities. Thus, an incubation mixture volume chosen for a 96well plate may exceed the volume capacity that can be accommodated in ahigher density format. It is convenient to scale the incubation mixturethrough these various formats by relating the amount of paramagneticmetal ion delivered to the well to the volume of incubation mixture.Thus, a more concentrated paramagnetic metal ion reagent may be utilizedin higher density plate format. Having the foregoing in mind, the amountof paramagnetic metal ion added to the incubation mixture can be basedon the volume of the incubation mixture when it is desired to use theassay in multiple plate formats. This is illustrated in Table 1 below.

In other embodiments of the present invention, homogenous aqueous assaysalso can be accomplished with the labeled enzyme substrate orparamagnetic metal ion affixed to a solid phase, rather than completelysoluble in solution. Examples of solid phases include polystyreneplates, membranes, or glass.

In practicing the present invention, the excitation and emission filtersused in measuring fluorescence are selected to provide wavelengths thatfall within the excitation/emission spectrum of the fluorophoreincorporated on the peptide substrate. In the case of LissamineRhodamine, a 560/590 filter excitation/emission filter can be utilized;the 560 filter exciting near the peak absorbance of the dye. When theassay is practiced at high peptide concentrations that result insubstantial self quenching, it is preferred to utilize off-peakillumination to limit inner filter effects and thereby increase assaydetection sensitivity. In all cases, a requirement for choice ofexcitation/emission filters utilized in the assay is that the selectedcombinations not present conditions where the photomultiplier tube issaturated in excess of its capacity for measurement. The fluorometergain setting is preferably set to allow the largest signal distinctionbetween the experimental sample and that of the reference.

While the invention has been described above with respect to assays ofthe activity of kinase, phosphatase and protease with peptidesubstrates, the invention is applicable to the assay of other enzymesrelying on substrates other than peptides. As to useful enzymes, theyare capable of introducing or removing a target group on a fluorecentlylabeled substrate or endproduct, and where the target group has theability to bind to a paramagnetic metal ion. Other enzymes includephospholipases that rely on phospholipid substrates, for example. Othersubstrates can include, in addition to phospholipids, proteins andcyclic nucleotides, among others.

While the assays described herein use a single fluorophore, it isrecognized that a plurality of fluorophores may also be incorporated.The plurality may be used to enhance the effective Stokes shift of thefluorophore excitation/emission spectral separation or be used toincrease the effective detection sensitivity of the labeled substrate orendproduct.

The following examples illustrate the invention.

EXAMPLE I Preparation of Working Solution Containing the ParamagneticMetal Ion as Fe⁺⁺⁺

9 volumes of Reagent A (0.555 M MES, 44.4 mM iminodiacetic acid, sodiumsalt, pH 5.8) and 1 volume of Reagent B (200 mM FeCl₃ in water) aremixed to give a final Working Solution containing 20 mM Fe³⁺, 40 mMiminodiacetic acid, disodium salt, 0.5 M MES (morphilinoethane sulfonicacid buffer) with a final pH of approximately 5.5. This reagent can befurther diluted with water to yield 0.5× strength working solution ordiluted to 0.25× strength working solution with water, for example. Theworking solution exhibits a time-dependent color change upon mixing.Visually this appears as a darkening of the solution to a reddish browncoloration. The working solution may be utilized immediately in theassay, or may be prepared a day before. Alternatively, the workingsolution may be briefly warmed to accelerate the color change to asteady state, and then allowed to cool to room temperature prior to use.

Table 1 illustrates use of the working solutions where a commonincubation mixture is directly scaled to accommodate a variety of platedensity formats. The amount of Fe⁺⁺⁺ to incubation mixture is heldconstant across these possible permutations with respect to the volumeof incubations. It can be seen from this table that the amount ofbuffering agent is also held constant with respect to the volume ofincubation mixture across these possible permutations. This formatallows for the convenience of moving the assay from one plate format toanother while ensuring delivery of sufficient Fe⁺⁺⁺ reagent andequivalent buffering capacity to maintain final pH consistency. Theassay is operational under all the illustrated formats. It can be seenfrom this table that the assay is flexible with respect to its practice.Preferably, the final well volumes are maximized to limit potentialinner filter effects that can occur when higher fluorophore-labeledpeptide substrates are utilized in the incubation mixture. TABLE 1 96Well 384 Well 1536 Well Plate Plate Plate Incubation mixture  30 18 3volume, ul/well/assay Working Solution 120 (0.25 × 36 (0.5 × 3 (1 ×Addition, ul/well/assay Fe³⁺ WS) Fe³⁺ WS) Fe³⁺ WS) Final Volume/well 150ul 54 ul 6 ul

EXAMPLE II Fluorescent Measurements

Fluorescent measurements were generally obtained using a BMG PolarStarfluorometric plate reader (BMG Labtechnologies Inc, Durham, N.C.) usinga 560/590 filter set, unless otherwise specified. A Tecan Sapphire platereader (Austria) with a 560/590 excitation/emission and 5 nm bandpass toachieve similar results.

EXAMPLE III Enzymatic Reaction Conditions for Kinase Enzymes

PKC (protein kinase C) assays were carried out in a reaction mixtureconsisting of 20 mM HEPES, 5 mM CaCl₂, 5 mM MgCl₂, 1 mM ATP (disodiumsalt), 1 mM DTT (dithiothreitol), 0.2 mg/ml phosphatidyl-L-serine, pH7.4. The enzyme preparation (purified PKC containing alpha, beta, andgamma isoforms, Pierce Biotechnology, Rockford, Ill.) was diluted in 20mM HEPES, 0.05% Triton X-100, pH 7.4, immediately before use in theassay. The enzyme substrates used were either Myelin Basic ProteinPeptide 4-14 (EKRPSQRSKYL) or Glycogen Synthase Peptide (PLSRTLSVAAKK)or Pseudosubstrate Peptide (RFARKGSLRQKNV) labeled on the N-terminalamine with Lissamine Rhodamine.

PKA (protein kinase A) assays were carried out in a reaction mixtureconsisting of 20 mM HEPES, 0.1 mM cAMP, 5 mM MgCl₂, 1 mM ATP (disodiumsalt), 1 mM DTT, pH 7.4. The enzyme preparation (PKA catalytic unit,Promega, Madison, Wis.) was diluted in 20 mM HEPES, 0.05% Triton X-100,pH 7.4, immediately before use in the assay. The enzyme substrate wasKemptide having a sequence of LRRASLG and labeled on the N-terminalamine Lissamine Rhodamine.

Tyrosine kinase (Src p60c-src, partially purified, UpstateBiotechnology, Waltham, Mass.) assays were carried out in a reactionmixture consisting of 20 mM HEPES, 5 mM MgCl₂, 1 mM ATP (disodium salt),1 mM DTT, pH 7.4. The enzyme substrate Tyrosine Kinase Peptide having asequence of KVEKIGEGTYGVVYK and labeled on the N-terminal alpha aminewith Lissamine Rhodamine.

Enzyme activity for PKC, PKA and tyrosine kinase is stated in mU(milliUnits), where 1 mU of transfers 1 pmole of phosphate per minuteunder conditions determined by the vendor.

EXAMPLE IV Detection of Differential Phosphorylation

Enzyme reactions were conducted according to Example III, and followingenzymatic reaction, the Working Solution prepared according to Example Iwas added, thus stopping the reaction. The plates were then measured forfluorescent signal. FIGS. 1-5 illustrate the results.

The following Examples, V and VI, illustrate the assay system of thepresent invention in assaying differential kinase enzymatic activity inthe presence of inhibitors.

EXAMPLE V Staurosporine PKC Inhibition

Staurosporine, a known ATP-site protein kinase inhibitor, was titratedout in triplicate in 10% DMSO over 22 wells in black 384-wellpolystyrene Labsystems plates. PKC enzyme and Lissamine Rhodaminelabeled Pseudosubstrate peptide were added to a final concentration of0.04 Units and 60 μM, respectively, in a final reaction concentration of20 mM HEPES, pH 7.4, 1.0 mM CaCl₂, 5 mM MgCl₂, 1 mM DTT, and 0.2 mgphosphatidyl-L-serine. 10 μM ATP was added to start the reaction. Thefinal incubation mixture in the well was 18 μl. The reaction was stoppedat 90 minutes with the addition of the Working Solution preparedaccording to Example I and then diluted with 10 volumes of water.Fluorescence was measured using a BMG FluoStar plate reader with a544/590 ex/em filter set. The results are illustrated in FIG. 6. Thecalculated IC₅₀ was found to be 0.8 nM.

EXAMPLE VI PKI, PKA Inhibition

PKI, a known synthetic peptide inhibitor of PKA, was titrated out intriplicate in 0.1 mg/ml BSA over 14 wells in black 384-well polystyreneLabsystems plates. PKA enzyme and Lissamine Rhodamine-labeled Kemptidesubstrate were added to a final concentration of 0.02 Units and 60 μM,respectively, in a final reaction concentration of 20 mM HEPES, pH 7.4,5 mM MgCl₂, and 1 mM DTT. 1.0 mM ATP was added to start the reaction.The reaction was stopped at 90 minutes with the addition of the WorkingSolution prepared according to Example I and then diluted with 10volumes of water. Fluorescence was measured using a BMG FluoStar platereader with a 544/590 ex/em filter set. The results are illustrated inFIG. 7. The calculated IC₅₀ was 2.6 nM.

EXAMPLE VII Enzymatic Reaction Conditions for Phosphatase Enzymes

PTP-Beta and PTP-1B (commercially available protein tyrosinephosphatases) assays were carried out in a reaction mixture consistingof 20 mM HEPES, 1.5 mM DTT, and 0,5 mM EDTA, pH 7.4. The enzymesubstrate used for both enzymes was Lissamine Rhodamine labeled on theN-terminal amine of the following phosphotyrosine peptide substrate(KVEKIGEGTpYGVVYK).

A PP2A (protein serine/threonine phosphatase) assay was carried out in areaction mixture consisting of 20 mM HEPES, 1.5 mM DTT, pH 7.4. Theenzyme substrate was Lissamine Rhodamine labeled on the N-terminal amineof phospho Kemptide having a sequence of LRRApSLG.

EXAMPLE VIII Detection of Differential Dephosphorylation

Enzyme reactions were conducted according to Example VII, and followingenzymatic reaction, the Working Solution prepared according to Example Iwas added. The plates were then measured for fluorescent signal. FIGS. 8and 9 illustrate the results.

The following Examples, 1× and X, illustrate the assay system of thepresent invention in assaying differential phosphatase enzymaticactivity in the presence of inhibitors.

EXAMPLE IX Sodium Ortho Vanadate, PTP-1B Phosphatase Inhibition

Sodium Ortho Vanadate, a known general phosphatase inhibitor, wastitrated in triplicate in 20 mM HEPES, pH 7.4, over 24 wells in a white96-well polystyrene Coster plate. PTP-1B phosphatase enzyme andLissamine Rhodamine labeled PhosphoTyrosine peptide substrate in ExampleVII were added to a final concentration of 22 mUnits and 30 μM,respectively, in a final reaction mixture of 20 mM HEPES. The finalincubation mixture in the well was 30 μl. The reaction was stopped at 60minutes with the addition of the Working Solution prepared according toExample I and then diluted with 30 volumes of water. Fluorescence wasmeasured using a BMG FluoStar plate reader with a 560/590 ex/em filterset. The results are illustrated in FIG. 11. The calculated IC₅₀ wasfound to be 1.0 μM.

EXAMPLE X Okadaic Acid/PP2A Phosphatase Inhibition

Okadaic Acid, another phosphatase inhibitor, solubilized in DMSO, wastitrated in triplicate in phosphatase dilution buffer over 24 wells inwhite 96-well polystyrene Costar plate. PP2A Phosphatase enzyme andLissamine Rhodamine labeled phospho Kemptide peptide substrate inExample VII were added to a final concentration of 22 milli Units and 30μM, respectively. The final incubation mixture in the well was 30 μl.The reaction was stopped at 60 minutes with the addition of the WorkingSolution prepared according to Example I and then diluted with 30volumes of water. Fluorescence was measured using a BMG FluoStar platereader with a 560/590 ex/em filter set. The results are illustrated inFIG. 10. The calculated IC₅₀ was found to be 1.5 nM.

EXAMPLE XI Enzymatic Reaction Conditions for Protease Enzymes

The protease, Trypsin (TPCK treated), from Pierce Biotechnology, Inc.,which cleaves peptide substrates C-terminal of arginine and lysineresidues, was assayed (BAEE units/mg protein) in a reaction mixtureconsisting of 25 mM TRIS, 150 mM NaCl, pH 7.2. The enzyme substratesused were Lissamine Rhodamine labeled on the N-terminal amine of eitherphosphoKemptide having a sequence of LRRApSLG or a longer derivativethereof having the sequence AGLARAGLALARLALALRRApSL.

EXAMPLE XII Detection of Differential Cleavage

Enzyme reactions were conducted according to Example XI, and followingenzymatic reaction, the Working Solution prepared in accordance withExample I was added. The plates were then measured for fluorescentsignal. FIGS. 12 and 13 illustrate the results.

EXAMPLE XIII Enzymatic Assays Using Fluorescein and Oregon GreenFluorophore Labels

PKA was diluted in 20 mM HEPES, pH 7.4, w/0.05% TX-100 and incubated ina final reaction buffer consisting of 20 mM HEPES, pH 7.4, 0.1 mM cAMP,1 mM ATP, 5 mM MgCl₂, 1 mM DTT with either fluorescein Kemptide (71.25uM) or Oregon Green Kemptide as the enzymatic substrate at a finalconcentration of 71.25 or 114.25 μM, respectively, with a final reactionvolume of 30 μl, for a reaction time of 1 hour in a 96 well plate formatusing an opaque white plate. Subsequently, 120 μl of 0.25×Fe(III)working reagent prepared according to Example I was added to theenzymatic reaction mixture and the fluorescence was measured using a485/538 ex/em filter set. The results are shown in FIGS. 14 and 15.

EXAMPLE XIV Time-Dependent Enzymatic Protein Phosphatase Activity Assays

IQ™ Assay of the Ser/Thr protein phosphatases PP-2Ca and PP2A. Thephosphatases were incubated with Lissamine Rhodamine-labeledphosphorylated Kemptide as the enzyme substrate. At each time point, theenzyme reaction mixture (10 μl) was stopped by, in place of thatoutlined in Table 1, the addition of 100 μl 0.25× Working Solution in a96 well white plate and fluorescence was measured, with a 10 μl portionof a no enzyme reaction mixture control treated in the same fashion ateach time point. The amount of enzyme utilized was 6.7 mU or 20 mU/10 μlof enzyme reaction mix for PP-2Ca and PP2A, respectively. Thefluorescence was then measured with a 560/590 filter set. The observedfluorescence increased with increasing dephosphorylation of thesubstrate. The results are shown in FIG. 16.

The disclosures of the patents and other references identified hereinare incorporated herein in their entirety.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventor for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventor expects skilled artisans to employ such variations asappropriate, and the inventor intends for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A composition formed in a fluorescence quench-based homogenous assay for enzymatic activity comprising a paramagnetic metal ion and a substrate for an enzyme or an enzymatic endproduct resulting from reaction of the enzyme with a substrate, the substrate or endproduct containing a fluorophore label and containing a target group to which the paramagnetic metal ion is bound to form a complex of the target and ion, said complex being in proximity to the fluorophore to cause the specific quenching of the fluorescence of the label when the complex forms.
 2. The composition of claim 1 wherein the target group is a phosphoryl group.
 3. The compositoin of claim 1 wherein the target group is an an imidazole group.
 4. The composition of claim 1 wherein the paramagnetic metal ion is Fe (III).
 5. The composition of claim 1 wherein the paramagnetic metal ion is Ni (II).
 6. The composition of claim 2 wherein the paramagnetic metal ion is Fe (III).
 7. The composition of claim 3 wherein the paramagnetic metal ion is Ni (II).
 8. The composition of claim 1 wherein the substrate or endproduct contains a single fluorophore label which is the only dye entity attached thereto.
 9. The composition of claim 6 wherein the substrate or endproduct contains a single fluorophore label which is the only dye entity attached thereto
 10. The composition of claim 7 wherein the substrate or endproduct contains a single fluorophore label which is the only dye entity attached thereto.
 11. The composition of claim 1 wherein the paramagnetic metal ion, in addition to being bound to be the target group, is coordinated with a chelator.
 12. The composition of claim 6 wherein the paramagnetic metal ion, in addition to being bound to be the target group, is coordinated with a chelator.
 13. The composition of claim 7 wherein the paramagnetic metal ion, in addition to being bound to be the target group, is coordinated with a chelator.
 14. A method for assaying the activity of an enzyme by contacting the enzyme with a population of fluorophore labeled substrate in an aqueous enzymatic reaction mixture, allowing the enzymatic reaction to proceed, contacting this reaction mixture with a paramagnetic metal ion to form a complex of the paramagnetic metal ion with a target group, said complex when in proximity to the fluorophore causing the specific quenching of the fluorescence from the fluorophore, measuring the intensity of the observed fluorescent emission from the mixture, relating the observed fluorescence from the mixture to that of an external reference, and ascribing a differential fluorescent signal, if any, between the two, the ascribed differential fluorescent signal of the sample being indicative of the final state of the fluorophore labeled substrate population after enzymatic reaction, and in turn an indicator of enzymatic activity.
 15. The method of claim 14 wherein the enzyme whose activity is being assayed is a kinase.
 16. The method of claim 14 wherein the enzyme whose activity is being assayed is a phosphatase.
 17. The method of claim 15 wherein the paramagnetic metal ion is Fe (III) and the target group is a phosphoryl group.
 18. The method of claim 16 wherein the paramagnetic metal ion is Fe (III) and the target group is a phosphoryl group.
 19. The method of claim 14 wherein the substrate or endproduct contains a single fluorophore label which is the only dye entity attached thereto.
 20. The method of claim 15 wherein the substrate or endproduct contains a single fluorophore label which is the only dye entity attached thereto.
 21. The method of claim 16 wherein the substrate or endproduct contains a single fluorophore label which is the only dye entity attached thereto.
 22. The method of claim 14 wherein the paramagnetic metal ion, in addition to being bound to be the target group, is coordinated with a chelator.
 23. A kit comprising a paramagnetic metal ion and an instruction booklet referencing and/or describing the manner in which the assay can be accomplished with respect to one or more enzymes as set forth herein.
 24. The kit of claim 23, further including a synthetic calibrator. 